The present invention relates to a method for quantifying protein tyrosine phosphatase (referred as PTP hereinafter) in biosamples, precisely a diagnostic method for disease by quantifying PTP using mass spectrometry and profiling of comparative PTP levels. By quantifying PTP in biosamples and profiling thereof according to the method of the present invention, disease can be diagnosed and diverse disease conditions and health conditions can be confirmed via profiling.
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7. A method for quantification of protein tyrosine phosphatase (PTP) comprising the following steps:
1) concentrating PTP in a sample separated from a test subject;
2) hydrolyzing the concentrated sample of step 1);
3) adding a known amount of an isotope-substituted synthetic standard peptide to the hydrolyzed sample of step 2), wherein the synthetic standard peptide is selected from SEQ ID NOs:191, 220, 225, 236, 246, 202, 231, 186, 172, 176 and 238; and
4) comparing the levels of wild type peptide and the isotope-substituted synthetic standard peptide of step 3) to calculate a quantity of the wild type peptide expression.
1. A method for quantification of protein tyrosine phosphatase (PTP) comprising the following steps:
1) hydrolyzing a sample separated from a test subject;
2) adding a known amount of an isotope-substituted synthetic standard peptide to the hydrolyzed sample of step 1), wherein the synthetic standard peptide is selected from SEQ ID NOs:191, 220, 225, 236, 246, 202, 231, 186, 172, 176 and 238;
3) extracting wild type peptide and the isotope-substituted synthetic standard peptide from the hydrolyzed sample of step 2), followed by quantitative analysis; and
4) comparing the levels of the wild type peptide and the isotope-substituted synthetic standard peptide of step 3) to calculate a quantity of the wild type peptide expression.
9. A screening method of a colon cancer, liver cancer, or lung cancer related biomarker comprising the following steps:
1) hydrolyzing a sample separated from a subject with colon cancer, liver cancer, or lung cancer;
2) adding a known amount of an isotope-substituted synthetic standard peptide to the hydrolyzed sample of step 1), wherein the synthetic standard peptide is selected from SEQ ID NOs:191, 220, 225, 236, 246, 202, 231, 186, 172, 176 and 238;
3) extracting the wild type peptide and the isotope-substituted synthetic standard peptide from the hydrolyzed sample of step 2), followed by quantitative analysis thereof;
4) comparing the levels of a wild type peptide and the isotope-substituted synthetic standard peptide of step 3) to calculate a quantity of the wild type peptide expression; and
5) comparing the quantity of the wild type peptide of step 4) and the quantity of the wild type peptide extracted from a normal subject to confirm the standard peptide demonstrating a significant difference.
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This is the U.S. National Stage of International Application No. PCT/KR2008/004541, filed Aug. 5, 2008, which was published in English under PCT Article 21(2), which in turn claims the benefit of Korean Patent Application No 10-2007-0125161, filed Dec. 4, 2007.
The present invention relates to a method for quantifying protein tyrosine phosphatase (referred as PTP hereinafter) in biosamples.
Protein tyrosine phosphorylation-dephosphorylation plays a very important role in intracellular signal transduction system. In particular, protein tyrosine phosphorylation-dephosphorylation is involved in changes of cells such as responses to foreign stimuli, cell growth, differentiation and apoptosis, etc. Therefore, protein tyrosine kinase (PTK; Curr Pharm Des 13:2751-65, 2007; Curr Med Chem 14:2214-34, 2007) and protein tyrosine phosphatase (PTP) are important target proteins for the treatment of such diseases accompanying the change of cells as cancer, vascular disease, immune disease and nervous disease (Curr Cancer Drug Targets 6:519-532, 2006; Med Res Rev 27:553-73, 2007). Human has approximately 100 kinds of PTPs (Cell 117:699-711, 2004). 20 kinds of these PTPs have been confirmed to be related to disease so that they have been targets of the development of a novel drug. And the remaining 80 kinds of PTPs are presumed to be related to disease as well.
According to the previous reports, PTP levels vary from disease and cell conditions (Crit Rev Oncol/Hemat 52:9-17, 2004; Expert Opin Therapeutic Targets 10:157-177, 2006). However, since there is no tools to measure the level of PTP in cells or blood directly, indirect methods such as measuring intracellular mRNA level by RT-PCR or Western blotting using commercial PTP antibody against limited PTP proteins are being used to quantify PTP. However, quantifying mRNA cannot tell exact amount of PTP. Besides, mRNA measurement is not possible with blood or urine samples. In the case of Western blotting, precise quantification of PTP is still difficult because only 10 PTP antibodies have been known and sensitivity of these antibodies is not very good. Despite PTPs are highly potent as a biomarker, development of a method for diagnosis of disease using these excellent biomarkers is not advanced, yet.
Blood samples, among many biosamples, are excellent test samples for diagnosis of disease using a biomarker, because of easiness in sampling and diversity of materials included in blood. Blood circulates everywhere in human body, during which blood takes cells a bit from each and every part of the body. These cells are broken, so that proteins included in those cells are flowing into blood. So, blood contains such proteins, telling conditions of the body. However, the amounts of such blood proteins are very small, so the presence of blood protein itself is sometimes neglected. In the meantime, large amount of proteins such as albumin and immunoglobulin are included in blood, which make it difficult to analyze minute proteins derived from cell.
To measure those PTPs existing at femto or atto mole level in blood, the present inventors selected standard peptides of PTP active domain facilitating the analysis of 80 kinds of PTPs by using mass spectrometer. So, peptides collected with antibodies binding specifically to the standard peptides are quantified by SISCAPA (Stable Isotope Standards and Capture by Anti-peptide Antibodies) technique that is a method to quantify protein based on mass spectrometry (Mol Cell Proteomics 5:573-588, 2006); Proc Natl Acad Sci USA 100:6940-6945, 2003). As a result, several PTPs demonstrated different levels between normal individual and cancer patient. The present inventors further completed this invention by confirming that the method of the invention facilitating analysis by PTP panel constructed by using standard peptides and their antibodies can be effectively used for diagnosis of disease.
It is an object of the present invention to provide a standard peptide derived from protein tyrosine phosphatase (PTP) for quantitative analysis of PTP.
It is another object of the present invention to provide an antibody binding specifically to the standard peptide for quantitative analysis
It is also an object of the present invention to provide a method for quantification of PTP in sample using the standard peptide and the antibody.
It is further an object of the present invention to provide a screening method of a cancer related biomarker using the standard peptide and the antibody.
It is also an object of the present invention to provide a screening method of a specific disease related biomarker using the standard peptide and the antibody.
It is also an object of the present invention to provide a method for diagnosis of cancer using the standard peptide and the antibody.
It is also an object of the present invention to provide a diagnostic kit for disease containing an antibody binding specifically to the standard peptide of the biomarker screened by the specific disease related biomarker screening method.
It is also an object of the present invention to provide a use of the synthetic standard peptide for quantification of PTP
It is also an object of the present invention to provide a use of the synthetic standard peptide for the screening of a cancer-related biomarker.
It is also an object of the present invention to provide a use of the synthetic standard peptide for the screening of a specific disease related biomarker.
To achieve the above objects, the present invention provides a standard peptide for quantitative analysis of PTP expressed in the sample which is produced by hydrolysis of protein tyrosine phosphatase (PTP) having PTP active domain comprising the amino acid sequences represented by SEQ. ID. NO: 113-NO: 168 and the amino acid sequences represented by SEQ. ID. NO: 256-NO: 260 and SEQ. ID. NO: 271-NO: 290.
The present invention also provides a synthetic standard peptide for quantitative analysis of PTP expression which has the amino acid sequence selected from the sequences represented by SEQ. ID. NO: 169-NO: 255.
The present invention further provides an antibody binding specifically to the standard peptide or the synthetic standard peptide.
The present invention also provides a method for quantification of PTP comprising the following steps:
1) hydrolyzing a sample separated from a test subject;
2) adding an isotope-substituted synthetic standard peptide to the hydrolyzed sample of step 1);
3) extracting the wild type peptide and the isotope-substituted synthetic standard peptide from the hydrolyzed sample of step 2), followed by quantitative analysis; and
4) comparing the levels of the wild type peptide and the isotope-substituted synthetic standard peptide of step 3) to calculate absolute quantity of the wild type peptide expression.
The present invention also provides a method for quantification of PTP comprising the following steps:
1) concentrating PTP in a sample separated from a test subject;
2) hydrolyzing the concentrated sample of step 1);
3) adding an isotope-substituted synthetic standard peptide to the hydrolyzed sample of step 2); and
4) comparing the levels of the wild type peptide and the isotope-substituted synthetic standard peptide of step 3) to calculate absolute quantity of the wild type peptide expression.
The present invention also provides a screening method of a cancer related biomarker comprising the following steps:
1) hydrolyzing a sample separated from a subject with cancer;
2) adding an isotope-substituted synthetic standard peptide to the hydrolyzed sample of step 1);
3) extracting the wild type peptide and the isotope-substituted synthetic standard peptide from the hydrolyzed sample of step 2), followed by quantitative analysis thereof;
4) comparing the levels of the wild type peptide and the isotope-substituted synthetic standard peptide of step 3) to calculate absolute quantity of the wild type peptide expression; and
5) comparing the absolute quantity of the wild type peptide of step 4) and the absolute quantity of the wild type peptide extracted from a normal subject to confirm the standard peptide demonstrating a significant difference.
The present invention also provides a screening method of a specific disease related biomarker comprising the following steps:
1) hydrolyzing a sample separated from a subject with a specific disease;
2) adding an isotope-substituted synthetic standard peptide to the hydrolyzed sample of step 1);
3) extracting the wild type peptide and the isotope-substituted synthetic standard peptide from the hydrolyzed sample of step 2), followed by quantitative analysis thereof;
4) comparing the levels of the wild type peptide and the isotope-substituted synthetic standard peptide of step 3) to calculate absolute quantity of the wild type peptide expression; and
5) comparing the absolute quantity of the wild type peptide of step 4) and the absolute quantity of the wild type peptide extracted from a normal subject to confirm the standard peptide demonstrating a significant difference.
The present invention also provides a method for diagnosis of cancer comprising the following steps:
1) hydrolyzing a sample separated from a subject with cancer;
2) adding a synthetic standard peptide substituted with an isotope of one or more biomarkers screened by the cancer related biomarker screening method to the hydrolyzed sample of step 1);
3) extracting the wild type peptide and the isotope-substituted synthetic standard peptide from the hydrolyzed sample of step 2), followed by quantitative analysis thereof;
4) comparing the levels of the wild type peptide and the isotope-substituted synthetic standard peptide of step 3) to calculate absolute quantity of the wild type peptide expression; and
5) comparing the absolute quantity of the wild type peptide of step 4) and the absolute quantity of the wild type peptide extracted from a normal subject to confirm the standard peptide demonstrating a significant difference.
The present invention also provides a method for diagnosis of cancer comprising the following steps;
1) concentrating PTP in a sample separated from a test subject;
2) hydrolyzing the concentrated sample of step 1);
3) adding a synthetic standard peptide substituted with an isotope of one or more biomarkers screened by the cancer related biomarker screening method to the hydrolyzed sample of step 2);
4) comparing the levels of the wild type peptide and the isotope-substituted synthetic standard peptide of step 3) to calculate absolute quantity of the wild type peptide expression; and
5) comparing the absolute quantity of the wild type peptide of step 4) and the absolute quantity of the wild type peptide extracted from a normal subject to confirm the standard peptide demonstrating a significant difference.
The present invention also provides a diagnostic kit for disease containing an antibody binding specifically to the standard peptide of the biomarker screened by the specific disease related biomarker screening method.
The present invention also provides a diagnostic kit for disease containing a primary monoclonal antibody binding specifically to a standard peptide of the biomarker screened by the specific disease related biomarker screening method and a secondary monoclonal antibody binding specifically to the overall region except the region where the primary monoclonal antibody is conjugated.
The present invention also provides a use of the synthetic standard peptide for quantification of PTP.
The present invention also provides a use of the synthetic standard peptide for the screening of a cancer-related biomarker.
In addition, the present invention provides a use of the synthetic standard peptide for the screening of a specific disease related biomarker.
Diverse disease conditions and health conditions can be confirmed by measuring and profiling PTP level in a biosample according to the method of the present invention. The method of the present invention can also be effectively used for prediction of prognosis after surgical operation and for determination of treatment strategy. In particular, the method of the present invention facilitates exact PTP quantification even with such a biosample containing a very small amount of PTP like blood, so that it can be effectively used for diagnosis of disease and screening of health condition with samples easily taken.
The application of the preferred embodiments of the present invention is best understood with reference to the accompanying drawings, wherein:
a: sequence of LMPTP active domain protein (residues 2-158 of SEQ ID NO: 164); and,
b: sequence of trypsin hydrolyzing peptide of LMPTP active domain (residues 2-158 of SEQ ID NO: 164).
SISCAPA (Stable Isotope Standards and Capture by Anti-peptide Antibodies): quantitative analysis method of peptides collected with antibodies based on mass spectrometry;
MRM (Multiple Reaction Monitoring): proteome analysis method using mass spectrometry to analyze complicated proteins and peptides in blood;
The Sequence Listing is submitted as an ASCII text file in the form of the file named Sequence_Listing.txt, which was created on May 31, 2010, and is 260,857 bytes, which is incorporated by reference herein.
Terms used in this invention are described hereinafter.
“Wild type peptide” indicates PTP peptide existing in the hydrolyzed sample of a test subject. In this invention, this peptide is a counter-part of a standard peptide labeled or substituted with a radio-isotope added to the hydrolyzed sample.
Hereinafter, the present invention is described in detail.
The present invention provides a standard peptide for quantitative analysis of PTP expressed in the sample which is produced by hydrolysis of protein tyrosine phosphatase (PTP) having PTP active domain comprising the amino acid sequences represented by SEQ. ID. NO: 113-NO: 168 and the amino acid sequences represented by SEQ. ID. NO: 256-NO: 260 and SEQ. ID. NO: 271-NO: 290.
In a preferred embodiment of the present invention, purified PTP active domain was hydrolyzed by trypsin to obtain PTP active domain peptide, followed by tandem mass spectrometry. As a result, 5-10 PTP specific peptides were obtained, among which the peptide that contained a residue replaceable with a stable isotope but not contained cysteine or methionine, the oxidation risk factors, and had high detection strength was selected as standard peptide. That is, considering the said conditions, standard peptide of PTP active domain was selected for quantitative analysis of PTP (see
The said standard peptide is composed of protein tyrosine phosphatase (PTP) active domain having the amino acid sequences represented by SEQ. ID. NO: 113-NO: 168 (1-56 of Table 1), PTP protein having the amino acid sequences represented by SEQ. ID. NO: 271-NO: 290 expressed by MBP fusion (described in Korean Patent No. 10-0746993) and a peptide appropriate for optimum ionization generated by hydrolyzing a protein having the amino acid sequences represented by SEQ. ID. NO: 256-NO: 260.
The present invention also provides a synthetic standard peptide for quantitative analysis of PTP expression which has the amino acid sequence selected from the sequences represented by SEQ. ID. NO: 169-NO: 255.
The synthetic standard peptide is composed of those peptides having a residue replaceable with an amino acid having a stable radio-isotope such as leucin or valine but not containing a residue having high risk of oxidation such as cysteine or methionine. The said replacement can be performed by adding an amino acid having a stable isotope during synthesis or labeling a specific amino acid with a functional group having a stable isotope after synthesis. In this invention, the radio-isotope is binding to the standard peptide in order to make mass different from that of the wild type peptide, which makes distinguishment between the two peptides easy. This radio-isotope is not necessarily included in inner-part of the standard peptide but instead it can be bound to OH-terminal of the standard peptide. In the standard peptide, any amino acid except those containing such a residue having risk of oxidation can be substituted with a stable isotope. The said stable isotope is selected from the group consisting of 13C, 15N and 2H. In a preferred embodiment of the present invention, 13C and 15N were used.
The present invention further provides an antibody binding specifically to the standard peptide or the synthetic standard peptide.
The antibody herein includes polyclonal or monoclonal antibody. Polyclonal antibody is used for the extraction of standard peptide from the hydrolyzed sample and quantitative analysis thereof, while monoclonal antibody is used for quantitative analysis of standard peptide in the sample, but not always limited thereto. In a preferred embodiment of the present invention, the polyclonal antibody was used to obtain the wild type standard peptide and the isotope-substituted standard peptide from serums of cancer patients and normal health people added with the isotope-substituted synthetic standard peptide after hydrolysis. Quantitative analysis was performed with the obtained wild type peptide and the isotope-substituted peptide using triple quadrupole analyzer. As a result, the wild type standard peptide of PTP T46 was quantified and absolute quantity of the wild type standard peptide was calculated by comparing with the peak of the isotope-substituted standard peptide (see
A polyclonal antibody can be prepared as follows; one of the said standard peptide of PTP active domains is injected into a test animal; blood sample is taken from the animal; and then serum containing antibody is separated to isolate the antibody. Such polyclonal antibody can be purified by any methods known to those in the art and can be produced from host animals which are exemplified by goat, rabbit, sheep, monkey, horse, pig, cow, dog, etc. A monoclonal antibody can be prepared by any method that facilitates the production of antibody molecules via culturing the continuous cell line. The method is exemplified by hybridoma technique, human-B-cell hybridoma technique, and EBV-hybridoma technique, but not always limited thereto (Kohler G et al., Nature 256:495-497, 1975; Kozbor D et al., J Immunol Methods 81:31-42, 1985; Cote R J et al., Proc Natl Acad Sci 80:2026-2030, 1983; Cole S P et al., Mol Cell Biol 62:109-120, 1984). An antibody fragment containing a specific binding site for one of the said standard peptide of PTP active domains can be prepared.
For example, F(ab′)2 fragment can be prepared by fractionation of an antibody molecule by using pepsin and Fab fragment can be prepared by reducing disulfide bridge of F(ab′)2 fragment, but not always limited thereto. Alternatively it is also possible to identify a monoclonal Fab fragment having desired specificity by constructing Fab expression library (Huse W D et al., Science 254: 1275-1281, 1989).
The present invention also provides a method for quantification of PTP comprising the following steps:
1) hydrolyzing a sample separated from a test subject;
2) adding an isotope-substituted synthetic standard peptide to the hydrolyzed sample of step 1);
3) extracting the wild type peptide and the isotope-substituted synthetic standard peptide from the hydrolyzed sample of step 2), followed by quantitative analysis; and
4) comparing the levels of the wild type peptide and the isotope-substituted synthetic standard peptide of step 3) to calculate absolute quantity of the wild type peptide expression.
The sample of step 1) comprise blood, tissue and exudate For the hydrolysis above, any protease that is capable of recognizing cleavage site of protein included in the sample to digest thereof can be used, and preferably trypsin, chymotrypsin, pepsin, thermolysis and proteinase K are selected. In a preferred embodiment of the present invention, trypsin was used.
The wild type peptide and the isotope-substituted synthetic standard peptide of step 3) are extracted by using a ligand or an antibody binding specifically to the said peptides. The antibody herein can be polyclonal antibody or monoclonal antibody, but polyclonal antibody is preferred to increase yield of extraction. In a preferred embodiment of the present invention, polyclonal antibody conjugated column was used. To obtain the standard peptide, PTP in the sample is hydrolyzed by trypsin and the obtained standard peptide is concentrated. Or, PTP as a protein is concentrated and then hydrolyzed by using trypsin. Particularly, almost every PTP has the same enzyme active site. Even if the structures of the enzyme active sites of different PTPs are a bit different, they have much in common such as active cysteine, etc. So, based on such homology, a low-molecular substance is designed to be conjugated to almost every PTP and then biotin or an analogue thereof is adhered to the low-molecular substance, which is going to be used for PTP concentration.
Quantitative analysis of step 3) is performed by LC/MS mass spectrometry, SELDI (Surface-Enchanced Laser Desorption/Ionization) and sandwich ELISA, but not always limited thereto.
In a preferred embodiment of the present invention, PTP panel composed of the standard peptide of PTP active domain was constructed and used for quantitative analysis of the standard peptide of patients with colon cancer, liver cancer and stomach cancer. As a result, 18 PTPs were detected in total. 10 out of the 18 PTPs were only detected in cancer patients but not in normal healthy people. The rest 8 PTPs were detected in normal healthy people as well but the levels of them in cancer patients were significantly higher, suggesting that they can be used for diagnosis of colon cancer, liver cancer and stomach cancer (see Table 5). Particularly, three PTPs (T46, pk32 and pk3) were able to be quantified. In the case of PTP T46 standard peptide, expression was slightly varied from individuals and types of cancer but regularly detected in general (see
The present invention also provides a method for quantification of PTP comprising the following steps:
1) concentrating PTP in a sample separated from a test subject;
2) hydrolyzing the concentrated sample of step 1);
3) adding an isotope-substituted synthetic standard peptide to the hydrolyzed sample of step 2); and
4) comparing the levels of the wild type peptide and the isotope-substituted synthetic standard peptide of step 3) to calculate absolute quantity of the wild type peptide expression.
If PTP is concentrated in sample before hydrolysis, analysis process can be simplified. PTP concentration in the sample of step 1) is performed by using a compound specifically binding to PTP enzyme active site. Almost every PTP has the same enzyme active site. Even if the structures of the enzyme active sites of different PTPs are a bit different, they have much in common such as active cysteine, etc. So, based on such similarity, a low-molecular substance is designed to be bound to almost every PTP and then biotin or an analogue thereof is adhered to the low-molecular substance, which is going to be used for PTP concentration.
The present invention also provides a screening method of a cancer related biomarker comprising the following steps:
1) hydrolyzing a sample separated from a subject with cancer;
2) adding an isotope-substituted synthetic standard peptide to the hydrolyzed sample of step 1);
3) extracting the wild type peptide and the isotope-substituted synthetic standard peptide from the hydrolyzed sample of step 2), followed by quantitative analysis thereof;
4) comparing the levels of the wild type peptide and the isotope-substituted synthetic standard peptide of step 3) to calculate absolute quantity of the wild type peptide expression; and
5) comparing the absolute quantity of the wild type peptide of step 4) and the absolute quantity of the wild type peptide extracted from a normal subject to confirm the standard peptide demonstrating a significant difference.
The present invention also provides a screening method of a specific disease related biomarker comprising the following steps:
1) hydrolyzing a sample separated from a subject with a specific disease;
2) adding an isotope-substituted synthetic standard peptide to the hydrolyzed sample of step 1);
3) extracting the wild type peptide and the isotope-substituted synthetic standard peptide from the hydrolyzed sample of step 2), followed by quantitative analysis thereof;
4) comparing the levels of the wild type peptide and the isotope-substituted synthetic standard peptide of step 3) to calculate absolute quantity of the wild type peptide expression; and
5) comparing the absolute quantity of the wild type peptide of step 4) and the absolute quantity of the wild type peptide extracted from a normal subject to confirm the standard peptide demonstrating a significant difference.
The present invention also provides a method for diagnosis of cancer comprising the following steps:
1) hydrolyzing a sample separated from a subject with cancer;
2) adding a synthetic standard peptide substituted with an isotope of one or more biomarkers screened by the cancer related biomarker screening method to the hydrolyzed sample of step 1);
3) extracting the wild type peptide and the isotope-substituted synthetic standard peptide from the hydrolyzed sample of step 2), followed by quantitative analysis thereof;
4) comparing the levels of the wild type peptide and the isotope-substituted synthetic standard peptide of step 3) to calculate absolute quantity of the wild type peptide expression; and
5) comparing the absolute quantity of the wild type peptide of step 4) and the absolute quantity of the wild type peptide extracted from a normal subject to confirm the standard peptide demonstrating a significant difference.
The cancer herein is selected from the group consisting of colon cancer, liver cancer and stomach cancer, but not always limited thereto.
The “significant difference” of step 5) indicates that the absolute quantity of the wild type standard peptide of a test subject is either higher or lower than that of a normal subject. Expressions of different standard peptides can vary from types of cancer and conditions thereof. Thus, cancer can be diagnosed by measuring the standard peptide level, either higher or lower. PTP proteins interact in cellular signal transduction pathway. So, unlike the conventional biomarkers, comparative amount of each PTP can be important information of biological functions. Therefore, comparison among expressions of tens of interacting PTPs can be a reliable diagnostic method that cannot be affected by diverse factors such as age, gender, lifestyle, etc.
The present invention also provides a method for diagnosis of cancer comprising the following steps;
1) concentrating PTP in a sample separated from a test subject;
2) hydrolyzing the concentrated sample of step 1);
3) adding a synthetic standard peptide substituted with an isotope of one or more biomarkers screened by the cancer related biomarker screening method to the hydrolyzed sample of step 2);
4) comparing the levels of the wild type peptide and the isotope-substituted synthetic standard peptide of step 3) to calculate absolute quantity of the wild type peptide expression; and
5) comparing the absolute quantity of the wild type peptide of step 4) and the absolute quantity of the wild type peptide extracted from a normal subject to confirm the standard peptide demonstrating a significant difference.
The present invention also provides a diagnostic kit for disease containing an antibody binding specifically to the standard peptide of the biomarker screened by the specific disease related biomarker screening method.
The antibody herein includes polyclonal antibody and monoclonal antibody. The kit herein additionally contains a secondary antibody labeled with an enzyme binding to the said antibody and reacting with a substrate for color development or a secondary antibody labeled with biotin. If a selected secondary antibody is labeled with an enzyme reactive to a substrate for color development, the substrate for color development and reaction buffer are additionally included.
The antibody can be fixed on a solid substrate. The solid substrate herein is selected from the group consisting of magnetic micro-bead, glass plate, bio-degradable organic polymer nano-particle such as PLGA and (micro)well plates.
The present invention also provides a diagnostic kit for disease containing a primary monoclonal antibody binding specifically to a standard peptide of the biomarker screened by the specific disease related biomarker screening method and a secondary monoclonal antibody binding specifically to the overall region except the region where the primary monoclonal antibody is conjugated.
The kit additionally contains a secondary antibody labeled with an enzyme binding to the secondary monoclonal antibody and reactive to a substrate for color development or a secondary antibody labeled with biotin. If a selected secondary antibody is labeled with an enzyme reactive to a substrate for color development, the substrate for color development and reaction buffer are additionally included.
The primary monoclonal antibody can be fixed on a solid substrate. The solid substrate herein is selected from the group consisting of magnetic micro-bead, glass plate, bio-degradable organic polymer nano-particle such as PLGA and (micro)well plates.
The present invention also provides a use of the synthetic standard peptide for quantification of PTP.
The present invention also provides a use of the synthetic standard peptide for the screening of a cancer-related biomarker.
In addition, the present invention provides a use of the synthetic standard peptide for the screening of a specific disease related biomarker.
Practical and presently preferred embodiments of the present invention are illustrative as shown in the following Examples.
However, it will be appreciated that those skilled in the art, on consideration of this disclosure, may make modifications and improvements within the spirit and scope of the present invention.
<1-1> Cloning of PTP Active Domain
Expression vectors capable of expressing 1-56 PTP active domains which have the amino acid sequences represented by SEQ. ID. NO: 113-NO: 1618 (Table 1) without help of a fusion protein were constructed.
The multiple cloning sites of PET28a (Novagen, USA) contains those restriction enzyme sites not included in DNA sequences of PTP active domains (SEQ. ID. NO: 113-SEQ. ID. NO: 168; Table 1) most, so that it was used as a backbone vector of the present invention.
As shown in Table 1, to amplify DNA sequences of PTP active domains 1-56 represented by SEQ. ID. NO: 113-SEQ. ID. NO: 168, PCR was performed with primers represented by SEQ. ID. NO: 1-SEQ. ID. NO: 112 using cDNA libraries of brain, muscle and testis purchased from Clontech as template DNAs as follows; at 95° C. for 5 minutes, at 95° C. for 1 minute, at 55-60° C. for 1 minute, at 72° C. for 90 seconds (30 cycles) and at 72° C. for 10 minutes. The amplified PCR products were digested with NdeI, EcoRI or BamHI, which were inserted into pET28a vector (Novagen, USA) and then named respectively pET28a-PTP 1-56.
TABLE 1
Nucleotide sequences of PTP active domain 1-56 and
primer sets
Amino acid location
(SEQ. ID. NO)
Forward primer
SEQ. ID.
No.
Name
DNA location
Reverse primer
NO
1
T4
225-793 (113)
CGCGACGCTAGCATGGCAGACGACAATAAGCTCTTC
1
673-2379
GCTGCGAAGCTTTACTTGAAGTTGGCATAATCTGA
2
2
T7
1684-1967 (114)
GGCACCCATATGCTAGTGGCTGTTGTTGCCTTATTG
3
5050-5901
GCGGGATCCTCAATGCCTTGAATAGACTGGATC
4
3
T48
1316-1897 (115)
GCCCCACATATGCGAGACCACCCACCCATCCCC
5
3946-5691
GGAAGATCTCTACGTTGCATAGTGGTCAAAGCTGCC
6
4
T8
821-1089(116)
GCGCCATATGGCAGACAAGTACCAGCAACTCTCCCTG
7
2461-3267
GCGCGGATCCCTCGGCTGGGGCCTGGGCTGACTGTTG
8
5
T23
1024-1335(117)
CCGTTACATATGGTGGAGAATTTTGAGGCCTACTTC
9
3070-4005
CCCGAATTCTTAGGCGATGTAACCATTGGTCTTTC
10
6
T39
879-1440(118)
CACATTGCTAGCATGAAGACATCAGACAGCTATGGG
11
2635-4320
CGGCTCAAGCTTCTAAGATGATTCCAGGTACTCCAA
12
7
T5
848-1452(119)
GCCCACCATATGGCCAGCGATACCAGCAGCCTG
13
2542-4356
GCGAGATCTTCAGCCAGAATTCAAGTATTCCAG
14
8
T38
709-979(120)
GACCGGCATATGCTTGCCAAGGAGTGGCAGGCCCTC
15
2125-2935
CCGGGATCCTCACTGGGGCAGGGCCTTGAGGAT
16
9
T12
674-1015(121)
CGCCAGCATATGGCCACGCGGCCACCAGACCGA
17
2020-3045
GCGGGATCCTCACTGGGGAAGGGCCTTGAGGAT
18
10
T15
851-1216(122)
GAGCATGCTAGCATGGCTAGGGAGTGTGGAGCTGGT
19
2551-3648
GCGGGATCCCTAGGACTTGCTAACATTCTCGTATAT
20
11
T10
327-650(123)
CCTTTCCATATGAAGCCCATAGGACTTCAAGAGAGAAG
21
979-1950
GACAGTAAGCTTTCAAAGTCTGCTCTCATACAGGCACA
22
12
T22
1367-1650(124)
CGCGAACATATGCTTAGCCACCCGCCAATTCCC
23
4099-4950
GGCGGATCCTCAGCCCACGGCCTCCAGCAGGGCCTC
24
13
T20
890-1180(125)
TTCGCTAGCGCCATCCGGGTGGCTGACTTG
25
2668-3540
GCGGGATCCCTAAAAGGAGCTTAAATATTCCAGTGCCA
26
14
PTP1B
1-299(126)
ATGGAGATGGAAAAGGAGTTCGAGCAGATC
27
1-897
GTCAACATGTGCGTGGCTACGGTCCTCACG
28
15
T25
1-387(127)
GCTCCCGCTAGCATGCCCACCATCGAGCGGGAG
29
1-1161
CGCGGATCCTTAGGTGTCTGTCAATCTTGGCCT
30
16
T41
157-537(128)
TCAGAGCATATGGAGGAGAAGATCGAGGATGAC
31
469-1611
GTGGACGCTAGCATGAAATATTTGGGCAGTCCCATT
32
17
T18
1-595(129)
GCCCCCCATATGGTGAGGTGGTTTCACCGAGAC
33
1-1785
CCGGAATTCTCACTTCCTCTTGAGGGAACCCTTG
34
18
pk32
63-360(130)
GAACCCCATATGTCTGTGAACACACCCCGGGAGGTC
35
187-1080
CGGGATCCTCAGGGGCTGGGTTCCTCAGGCAG
36
19
pk28
1-526(131)
CCGCGGCATATGGAACATCACGGGCAATTAAAA
37
1-1578
CGGGATCCTCACCTGCAGTGCACCACGACCGG
38
20
T32
2095-2490(132)
GCAGTACATATGAATGGGAAGTTATCAGAAGAG
39
6283-7468
GGCGGATCCTCACTTCAGAAGCTGAGGCTGCTGTTTTT
40
21
T40
866-1187(133)
GAGCAGCATATGGCAGGCCTGGAGGCACAGAAG
41
2596-3561
CGCGGATCCTTAAATGAGTCTGGAGTTTTGGAG
42
22
T2
839-1174(134)
CTAGGGCATATGAAAAAGACTCGAGTAGATGCA
43
2515-3522
CGCGGATCCTTAGATGAGCCTGGAGCTTTTCAG
44
23
pk4
173-323(135)
AGGCCGCATATGGTCATGGAAGTGGGCACCCTG
45
517-969
GGCGGATCCTCAGCTCCCAGCCTCTGCCGAACAG
46
24
pk7
174-338(136)
GTTCATATGAGTGCCACAGAGCCCTTGGAC
47
520-1012
GCGGGATCCTCAGGACGTGGCCAGCACCTGGGACTC
48
25
pk8
178-321(137)
GCGGACCATATGGGCCCAGTTGAAATCCTTCCCTTC
49
532-962
GCGAGATCTTCACGTGGAGGGCAGGATCTCAGATTCG
50
26
pk9
205-348(138)
GGCAGCCATATGTCCTTCCCAGTGGAGATCTTGCCC
51
613-1044
CGCGGATCCTCAGCTGAGTCCCAGCGTCCTCTCGAA
52
27
pk10
192-338(139)
GCTGGCCATATGTTGCGCCGCCTGCGCAAGGGC
53
574-1014
CGGGATCCTCACGTGGACTCCAGCGTATTGAG
54
28
T33
160-312(140)
TGCCCCCATATGGCTGGGGACCGGCTCCCGAGG
55
478-934
GCGGGATCCTCATGAGGGGGTGCCCGGGTCGCCCTG
56
29
pk12
201-351(141)
CGATCGCATATGGAGGGTCTGGGCCGCTCGTG
57
601-1053
CGGGATCCCTAGGTGGGGGCCAGCTCGAAGG
58
30
pk13
320-467(142)
CTGGACCATATGCAGCGGCTGAACATCGGCTAC
59
958-1401
CGGGATCCTCACACAACCGTCTCCACTCCCATC
60
31
T27
192-339(143)
GTTGCCCATATGGGGCCAACCCGAATTCTTC
61
574-1017
GGATCCTTATGATGCTCCAGTCTGGTTC
62
32
pk6
1-185(144)
GCCGCCCATATGTCGGGCTCGTTCGAGCTCTCG
63
1-555
CGGGATCCCTAGGGTTTCAACTTCCCCTCC
64
33
pk14
27-210(145)
GCCAAGCATATGGGCGGAAACCACATCCCCGAAAGG
65
79-628
GCGGGATCCTCAGGAATTCCAATTCTTTCTGATAGG
66
34
pk15
21-340(146)
AGCGCCCATATGGTCAGCTGTGCCGGGCAGATGCTG
67
61-1020
CGGGATCCTCATATTTTTCCTGTTTGTGATCC
68
35
pk33
1-188(147)
GGCTGGCATATGGCTGAGACCTCTCTCCCAGAG
69
1-564
CGGGATCCTCAGCTCTGGCCGGCACCCCGC
70
36
p44
1-198(148)
TCCCACCATATGGACTCACTGCAGAAGCAGGAC
71
1-601
GCCAAGGGTCAGGGATCCTGGCTG
72
37
p21
1-157(149)
CCCGGGCATATGGGCAATGGCATGACCAAGGTAC
73
1-471
GCGGGATCCTCACTTGCCGCCCTTGCGGGACAG
74
38
pk35
1-188(150)
GCGGGATCCTCACTTGCCGCCCTTGCGGGACAG
75
1-564
CGGGATCCTCACAGTGGAATCATCAAACGGAC
76
39
NE1
1-217(151)
CCAGGGGCTAGCCGCTAACTGGAAAGAAAA
77
1-651
GTCGGATCCTTAGCTTTCTTTGCCCTCTTG
78
40
p19
1-190(152)
ATGACAGCATCCGCGTCCTCCTTTTC
79
1-570
TTACATTGATATCATCATACGTAG
80
41
pk18
1-184(153)
GCAGCCCATATGGGGAATGGGATGAACAAGATC
81
1-552
CGGGATCCTTACAGTCTTCTGAGAAAGGCCCAG
82
42
p12
31-211(154)
GGGAAGCATATGGGTCGGGCGCACCGGGACTGG
83
91-603
GGCACCAAGCTTTCAGAACTCTTTAAGAACATCCAGCT
84
43
pk17
35-211(155)
CTGGAGCATATGCCAACCGTTCAACATCCTTTCC
85
103-633
GCGGGATCCTCATGCTTCCAGACCCTGCCGCAGC
86
44
p16
1-150(156)
GCGGCGGCTAGCATGGGCGTGCAGCCCCCCAACTTC
87
1-450
CGCGCCTCGAGTTTCGTTCGCTGGTAGAACTGGAA
88
45
T16
1-210(157)
GGCGGCGCTAGCATGGCTCACAACAAGATCCCGCCG
89
1-630
TGAGGATCCTTATGATTCCTTCTTTCCATCCTCATC
90
46
p18
306-450(158)
CCGGGACATATGGACAAGCCCTCCCTTATCTTC
91
916-1350
GCGGGATCCTCAGCTTGCATCCAAGATGCCTTC
92
47
NE3
306-350(159)
CTTGGTCATATGGATAGCCCTACACAGATATTTG
93
916-1350
GCGGGATCCTCACCTTGCCAGCAAGATCCCCTG
94
48
pk3
4-163(160)
GCGGCTCATATGAACCGCCCAGCTCCTGTGGAA
95
10-489
GCGGGATCCTCAGGAATCTTTGAAACGCAGCCGCAT
96
49
p49
14-167(161)
CGCCGAGCTAGCATGCGTTTTCTGATAACTCACAAC
97
40-501
CGGGATCCCTACTGAACACAGCAATGCCCATTG
98
50
p26
4-161(162)
GCGACCCATATGGCCCCGGTGGAGGTGAGCTACA
99
10-483
CGCGGATCCTCAGGTCTTGTGCGTGTGTGGGTCTTTG
100
51
T29
37-391(163)
GGCGGCCATATGTCGTCGACCTCGCCGGGTGTGAAG
101
109-1173
GCCGGATCCTTATTTGGAGAAGGCTGCTCTGTGTTGTC
102
52
T46
1-157(164)
ATGGCGGAACAGGCTACCAAGTCCGTG
103
1-471
TCAGTGGGCCTTCTCCAAGAACGCTCTGC
104
53
pk1
336-523(165)
GCTCTAGACTTATAGGAGACTTCTCCAAGGG
105
1006-1569
GCCCTAGGTCAGAGCTTCTTCAGACGACTGTAC
106
54
T47
378-566(166)
GACCACCATATGCTGATTGGAGATTACTCTAAGGCC
107
1132-1701
CCGGGATCCTCACTGGTCCTGCAGCCGGCTACA
108
55
T45
207-400(167)
GATTCTGCTAGCGGGCACCTGATTGGTGATTTTTCC
109
619-1200
CCGGGATCCTCATGGGCTCATGTCCTTCACCAG
110
56
Eya2
244-514(168)
GACAATCATATGGAGCGTGTGTTCGTGTGGGAC
111
730-1542
GAATTCTTATAAATACTCCAGCTCCAGGGCGTG
112
<1-2> Expression Vector for PTP Expressed by MBP Fusion
Vectors pET28a-MBP-PTP 1-5 for the expression of 5 PTPs via MBP fusion which have the amino acid sequences represented by SEQ. ID. NO: 256-NO: 260 (1-5 of Table 2) were constructed by the similar method to that described in Korean Patent No. 10-0746933. The primer sets used for the construction are shown in Table 2.
TABLE 2
Sequences of PTP 1-5 expressed as MBP fusion protein
and primer sets
Amino acid location
(SEQ. ID. NO)
Forward primer
SEQ. ID.
No
Name
DNA location
Reverse primer
NO
1
p45
1-295(256)
ATGAGGAGAACTTCCGGAGCAACC
261
1-885
CTATAGGCACGATGATACAAAATATAA
262
2
p46
149-420(257)
CCTCTAGCTAGCGATACGCGCAAAATTGTT
263
445-1260
GGATCCTTAATCCAAAGTCAGAAGTTTCC
264
3
p47
1-242(258)
CGCCCACATATGACAGCCATCATCAAAGAGAT
265
1-726
CGGGATCCTCAAAGTACATGAACTTGTCTTCC
266
4
T21
166-500(259)
CTTGCACATATGGGCTTTGACGTGCAGAACG
267
496-1500
CCGAGATCTTCATTGCACCAGTTTTACCAGGAA
268
5
T53
1-223(260)
TCGGCCCATATGCCTGGTTTGCTTTTATGTGAA
269
1-669
CGGAATTCTCAGTAGAGCGGATCCATGATG
270
<1-3> Conditions for Large Scale Expression with Maintaining Activity and Stability
E. coli was transfected respectively with the 56 vectors constructed in Example <1-1> and 5 vector constructed in Example <1-2> according to the method of Hanahan (Hanahan D, DNA Cloning vol. 109-135, IRS press 1985).
Particularly, E. coli BL21-DE3-RIL treated with CaCl2 was transfected with vectors constructed in Example <2-1> by heat-shock method. Then, the cells were cultured in medium containing kanamycin (Sigma, USA). Colonies having kanamycin resistance were selected. These colonies were cultured in LB medium for overnight and then some of the seed culture solution was inoculated in LB medium containing 30 μg/ml of kanamycin, followed by culture until stationary phase. The culture solution was diluted at the ratio of 1:100 and inoculated in fresh LB medium (400 ml/flask). Temperature was lowered slowly from 37° C. to 17° C. during 2-3 hour culture. Then, culture was continued at 17° C. at 200 rpm. When OD600 of the culture solution reached 0.5, IPTG was added at the lowest concentration (0.05-0.1 mM), followed by further culture for 20 or 16-18 hours to induce expression of PTP active domain.
<1-4> Conditions for Purification and Storage with Maintaining Activity and Stability
E. coli cultured in Example <1-3> was centrifuged at 4° C. at 6,000 rpm for 5 minutes. The cell precipitate was recovered, which was resuspended in 5 ml of cell lysis buffer (10 mM Tris-HCl buffer, pH 7.5, 10 mM EDTA). The cells were lysed using ultrasonicator at 4° C. Centrifugation was performed at 4° C. at 13,000 rpm for 10 minutes to separate supernatant and insoluble aggregate. Protein was eluted from the supernatant by linear density gradient using Ni—NTA resin (Qiagen, USA) at 4° C. for about 3 hours from low concentration buffer [20 mM Tris-HCl buffer, pH 7.5, 0.2 M NaCl, 1.0 mM PMSF, 4 mM β-mercaptoethanol (Sigma, USA)] to high concentration buffer [0.5 M imidazole (Sigma, USA) was added to the low concentration buffer]. The histidine tag of N-terminal of the eluted protein was eliminated by treating thrombin (protease) (Sigma, USA) by 1 unit/100 μg protein. The protein was purified by ion exchange chromatography (GE Healthcare, USA) and gel filtration chromatography (GE Healthcare, USA).
During the purification of PTP active domain, 10 mM β-mercaptoethanol (Sigma, USA) or DTT (Promega, USA) was added to the buffer and pH of the buffer was regulated to 6.5-8.0. The purified PTP active domain was stored at 4° C. with the addition of 10% glycerol in protein solution [10% glycerol solution prepared by adding 100-250 mM NaCl, 10 mM reducing agent (β-mercaptoethanol or DTT) and 0.5˜2 μg/ml protease inhibitor (Sigma, USA) to pH 7.5-8.0 Tris buffer].
<2-1> Hydrolysis of PTP Active Domain Using Trypsin
56 PTP active domains obtained by the method described in Example 1, 5 proteins expressed by MBP fusion and 20 proteins expressed by MBP fusion described in Korean Patent No. 10-0746933 and shown in Table 3 were hydrolyzed by using trypsin (Sequencing Grade Modified Trypsin; Promega, USA) protease. Particularly, denaturation of PTP active domains purified at the concentration of 1 mg/ml was performed with 50 mM Ammonium Bicarbonate 0.1% (w/v) Rapigest reagent (Waters, USA)/20 μl PTP active domain for 30 minutes at 60° C. After cooling at room temperature, 1 μg of trypsin was added thereto, followed by hydrolysis at 37° C. 2 hours later, the reaction was terminated by adding 0.5% (final conc.) TFA (trifluoroacetic acid; Burdick & Jackson Brand, USA). After incubation for 30 minutes at 37° C., the reaction mixture was centrifuged for 10 minutes at 13,000 rpm. Precipitate was eliminated and only hydrolyzed peptide solution was obtained. LC-MS/MS analysis was performed with the hydrolyzed peptide solution. For denaturation of the domain, urea, guanidine-HCL, etc can be used as a denaturant in addition to the Rapigest and heat treatment (90° C.) can also be accepted. Sample was loaded by on-line using trap column Symmetry C18 (Waters, USA) for nanoAcquity HPLC before analysis using Q-Tof premier mass spectrometer (Waters, USA). After loading, salts were eliminated, followed by drying under vacuum condition to give the sample for mass spectrometry.
TABLE 3
20 proteins expressed by MBP fusion described
in Korean Patent No. 10-9746993
Unitprot
Amino
SEQ.
accession
acid
ID.
No.
Name
Protein
no,
location
NO
1
p13
MTMR8
Q96EF0
122-704
271
2
p20
VHP(DUSP26)
Q05923
1-176
272
3
p24
TENC1
Q2NL80
125-320
273
4
pk16
MTMR7
Q9Y216
1-334
274
5
pk19
Laforin(EPM2A)
O95278
1-331
275
6
pk30
MKP6(DUSP14)
O95147
1-198
276
7
pk36
SSH3
Q8TE77
11-150
277
8
pk38
MK-STYX
Q9Y6J8
1-313
278
9
pk5
PAC-1(DUSP2)
Q05923
1-314
279
10
T1
PTP-PEST(PTPN12)
Q05209
1-324
280
11
T17
CD45(PTPRC)
Q5T5R0
465-1143
281
12
T19
MTMR3
Q13615
137-530
282
13
T24
MTMR1
Q13613
1-571
283
14
T26
RPTP δ (PTPRD)
P23468
1331-1912
284
15
T30
HD-PTP(PTPN23)
Q9H3S7
1060-1636
285
16
T3
PTP-HSCF(PTPN18)
Q99952
1-300
286
17
T31
PTPJ(PTPRU)
Q92729
817-1436
287
18
T35
LYP(PTPN22)
Q9Y2R2
1-326
288
19
T37
RPTP(PTPRE)
P23469
100-700
289
20
T6
RPTP γ (PTPRG)
P23470
825-1414
290
<2-2> Hydrolyzed Peptide Pattern and Ionization Pattern of Hydrolyzed PTP Active Domain
To determine peptide sequence of the hydrolyzed PTP active domain prepared in Example <2-1> and to record ionization pattern, Q-TOF premier/nanoAcquity (Waters, USA) system was used.
Peptides were separated from the proteins obtained in Example <2-1> by retention time on Atlantis C18 nanoAcquity column (Waters, USA) using density gradient method with solution A [0.1% formic acid (Fluka, Japan) deionized water] and solution B [0.1% formic acid acetonitrile (Fluka, Germany)] for 40 minutes at flow rate of 300 n2/min. At this time, ESI Source temperature was 80° C., and Capillary Voltage was maintained at 3.8 kV. MS spectrum of peptide detected on-line was analyzed, followed by MS/MS assay for maximum 10 seconds with peptide ions having +2 and +3 charges. MassLynx version 4.1 (Waters, USA) was used for MS/MS spectrum analysis. ProteinLynx v2.2 (Waters, USA) and Mascot release 2.1 (Matrix Science, USA) were used for peptide sequencing and hydrolyzed peptide analysis. By which, hydrolyzed peptide pattern and ionization tendency of each PTP active domain were analyzed.
<2-3> Selection and Synthesis of Standard Peptide of PTP Active Domain
Among the peptides confirmed to be efficiently ionized in Example <2-2>, the peptides which do not contain a residue having risk of oxidation such as cysteine or methionine but contain a residue replaceable with a stable isotope such as leucine or valine were selected. And each of those peptides was synthesized to 1-3 mg and to be replaced with an isotope.
Particularly, hydrolysis by trypsin resulted in cleavage of the region behind of Arg and Lys unless Pro is there, suggesting that the resultant peptide always includes C-terminal which is composed of Arg or Lys (
4 different FMOC amino acids (Cambridge Isotope Laboratories; CIL, USA), L-arginine-N—FMOC—13C6,15N4 (+10Da), L-lysine-α-N—FMOC—13C6,15N2 (+8Da), L-leucine-N—FMOC—13C6,15N(+7Da) and L-valine-N—FMOC—13C5,15N(+6Da), labeled with 13C and 15N were used to synthesize 1˜3 mg of isotope-substituted peptides according to Fmoc solid-phase synthesis method. Every trypsin hydrolase can be labeled with Arg or Lys of C-terminal, but when peptides contained Leu and Val in addition to C-terminal, those FMOC amino acids labeled with a stable isotope on Leu and Val were first selected because they were less expensive.
As a result, the peptide (or ion) having the amino acid sequences represented by SEQ. ID. NO: 169-NO: 255 generated by hydrolysis of PTP active domain was selected. Particularly, as shown in
TABLE 4
20 proteins expressed by MBP fusion described in
Korean Patent No. 10-9746993
Sequence of standard peptide; Mass and Energy value of optimal fragments
lab ID
Name
Sequence
Native
Eya2-1
Eya2
AVYVVIGDGVEEEQGAK*
881.95(+2)->weak
NE1-1
DUSP17, SKRP1
THILNVAYGVENAFLSDFTYK*
pep err
NE3-1
SSH2, slingshot2
EIDNFFPGV*FEYHNIR
666.32(+3)->616.32(+2)
p12-1
MOSP, DUSP23
IDPTVLLGALPL*R
689.43(+2)->852.57(+1)
p13-1
MTMR8
VPVLSYL*YK
361.22(+3)->weak
p16-R*
VHZ, DUSP25
FVQIVDEANAR*
631.33(+2)->774.37(+1)
p18-1
SSH1, slingshot1
EIDNFFPGL*FAYHNIR
651.66(+3)->594.32(+2)
p19-1
LMW-DSP21, DUSP21
SLFLSNGVAANDK*
668.35(+2)->875.42(+1)
p20-1
VHP
AAGAEEQL*AR
508.26(+2)->873.44(+1)
p21-1
VHY, DUSP15
DLDQL*GR
408.71(+2)->588.31(+1)
p24-1
C1-TEN
VATELQPSQR*
564.80(+2)->487.26(+1)
p44-1
TMDP, DUSP13B
QLQVL*DNR
493.28(+2)->517.27(+1)
p45-1
TENSIN
VLEFGWPDLHTPAL*EK
617.99(+3)->820.41(+2)
p46-1
TypPTP
VFLENYQILQYFIIR*
653.70(+3)->839.48(+1)
p47-1
PTEN
AQEALDFYGEV*R
466.56(+3) weak
pk10-1
PYST2, DUSP7
DSTNLDVL*GK
531.28(+2)->758.44(+1)
pk1-1
CDC25A
GYLFHTVAGK*
546.80(+2)->759.42(+1)
pk12-1
MKP-4, DUSP9
DSANLESL*AK
524.27(+2)->774.44(+1)
pk13-1
MKP-5, DUSP10
LNIGYVINVTTHLPL*YHYEK
796.43(+3)->1024.04(+2)
pk14-1
PIR1, DUSP11
IFTVGHQVPDDETIFK*
615.98(+3)->793.40(+2)
pk15-1
HYVH1, DUSP12
SSSIL*DHR
457.74(+2)->540.29(+1)
pk16-1
MTMR7
GYENEDNYSNIK*
482.54(+3)->weak
pk17-1
MGC1136
GTPEAYEGL*GIR
631.82(+2)->878.47(+1)
pk18-1
VHX, DUSP22
EEYGESPL*QDAEEAK
847.87(+2)->1000.50(+1)
pk19-1
Laforin, EPM2A
EPGGELSWEGNGPHHDR*
625.28(+3)->702.82(+2)
pk2-1
KAP, CDKN3
LAAHL*SSR
285.50(+3)->462.27(+1)
pk28-1
SHP2, PTPN11
FDSLTDLVEHYK*
489.58(+3)->602.81(+2)
pk30-1
MKP6, DUSP14
MVQTPYGIVPDV*YEK
580.30(+3)->750.36(+1)
pk32-1
HePTP, PTPN7
IPSNFVSPEDLDIPGHASK*
675.01(+3)->596.32(+1)
pk33-1
BEDP, DUSP13A
VDEVWPNL*FIGDAATANNR
701.35(+3)->737.38(+2)
pk35-1
DUSP20, LMW-DSP20
QPSVSGL*SQITK
622.85(+2)->613.84(+2)
pk36-1
SSH3, slingshot3
FTYHNV*R
312.83(+3)->388.23(+1)
pk38-1
STYX
IEDSPEAQILPFL*R
543.30(+3)->532.32(+1)
pk4-1
MKP-1, 3CH134
LDEAFEFV*K
549.28(+2)->869.44(+1)
pk5-1
PAC-1
LDEAFDFV*K
361.85(+3)->weak
pk6-2
VHR, T-DSP11
LGITHVLNAAEGR*
450.92(+3)->730.38(+1)
pk7-1
MKP-2, hVH2/TYP1
LEEAFEFV*K
556.29(+2)->869.44(+1)
pk8-1LK
hVH3/B23
LKEAFDYIK*
376.21(+3)->538.29(+1)
pk9-1
PYST1, MKP-3/rVH6
DSTNLDVL*EEFGIK
790.40(+2)->1049.55(+1)
PRL1-*KK
PRL1
FIEEL*KK
453.77(+2)->646.38(+1)
PRL12-R*
PRL1/2
YEDAVQFIR*
570.79(+2)->848.46(+1)
PRL2-*KK
PRL2
FTEEL*KK
447.75(+2)->646.38(+1)
PRL3-F*KK
PRL3
FLITHNIPTNATLSTFIEDL*KK
601.58(+4)->715.05(+3)
PRL3-R*
PRL3
YEDAIQFIR*
577.80(+2)->862.48(+1)
PTP1B-1
PTP1B
LHQEDNDYINASLIK*
591.63(+3)->645.39(+1)
PTPRT1
PTPRT
VTLIETEPLAEYV*IR
582.66(+3)->480.78(+2)
PTPRT2
PTPRT
GASTQNSNTV*EPEK
731.35(+2)->npp
SHP1-1
SHP1
DLSGLDAETL*LK
637.85(+2)->1046.57(+1)
SHP1-2
SHP1
GEPWTFL*VR
552.80(+2)->459.76(+2)
SHP1-3
SHP1
NQLL*GPDENAK
599.81(+2)->843.42(+1)
T10-1
PTP-SL, PCPTP
TILPNPL*SR
505.80(+2)->683.38(+1)
T1-1
PTP-PEST, PTP-P19
EQYELV*HR
358.52(+3)->408.72(+2)
T12-1
PTPRP, IA-2beta
SLAVL*TYDHSR
421.22(+3)->531.27(+2)
T15-1
GLEPP1, PTP-U2
FSLQFEEL*K
570.80(+2)->665.35(+1)
T16-R*
mRNA capping enzyme
YDSQVAEENR*
605.77(+2)->618.28(+1)
T17-1
CD45, LCA
YIAAQGPR*
438.24(+2)->599.33(+1)
T19-1
MTMR3, FYVE-DSP1
NADDEHLVQSV*AK
475.90(+3)->620.81(+2)
T2-1
PTPD1, PTP2E
HNTVTYGR*
316.50(+3)->weak
T21-1
MTRM4, FYVE-DSP2
SYTAAV*ANR
476.75(+2)->702.39(+1)
T22-1
RPTPsigma
TVDVYGHVTL*MR
464.24(+3)->weak
T23-1
DEP1, CD148
NIQTSESHPL*R
427.89(+3)->385.26(+1)
T24-1
MTMR1
VYDPV*SEYK
367.18(+3)->weak
T25-1
TCPTP, MPTP
EFEEL*DTQR
583.77(+2)->632.34(+1)
T26-1
RPTPdelta
PSDTTKYLLEQL*EK
555.63(+3)->759.43(+1)
T27-1
MKP-7, MKP-M
ILPNLYL*GCQR
430.57(+3)->weak
T29-1
CDC14B
NHNV*TTIIR
534.30(+2)->816.49(+1)
T30-1
HD-PTP, PTP-TD1
VLSL*QFR
288.18(+2)->npp
T3-1
PTP-HSCF, PTP20
YKDVLPYDQTR*
466.57(+3)->519.25(+1)
T31-1
PTPJ, PTP-U1
VADLLQHINQMK*
470.59(+3)->620.33(+2)
T32-1
PTP-BAS, FAP-1
VPLGDEGGYINASFIK*
560.63(+3)->679.38(+1)
T33-1
hVH5, M3/6, HB5
ILPHLYL*GSQK
423.58(+3)->521.79(+2)
T35-1
LYP, PEP
DGIIPENFSVFSL*IR
569.64(+3)->npp
T37-1
RPTP
DFLVTL*NQPQAR
467.92(+3)->nmatch
T38-1
IA-2
EIDIAATL*EHVR
456.25(+3)->562.81(+2)
T39-1
RPTPkappa
QNVVDVFHAV*K
419.23(+3)->601.35(+1)
T40-1
PTP36, PEZ, PTPD2
ANGIFSTAAL*PENAER
830.92(+2)->899.46(+1)
T4-1
RPTPalpha
AEGILDVFQTV*K
660.36(+2)->949.54(+1)
T41-1
STEP
APPLLHLV*R
339.22(+3)->424.28(+2)
T45-1
CDC25C
YVNPETVAALL*SGK
731.40(+2)->1085.62(+1)
T46-1
LMPTP
IELL*GSYDPQK
631.84(+2)->1020.54(+1)
T47-1
CDC25B
AFLLQTVDGK*
364.54(+3)->437.26(+2)
T48-1
LAR
NLYAHIQK*
493.78(+2)->759.42(+1)
T5-1
RPTPmu
TVDVFHAV*K
508.28(+2)->815.44(+1)
T53-1
STYX
SLSVHSGTTGSL*K
425.23(+3)->537.28(+2)
T6-1
RPTPgamma
HSDYINANYVDGYNK*
591.60(+3)->nmatch
T7-1
RPTPbeta
DSVDIYGAVHDL*R
487.24(+3)->579.80(+2)
T8-1
SAP1
TGTLIALDVLL*R
642.90(+2)->799.50(+1)
lab ID
SIS
QTOF
Quat
Eya2-1
885.95(+2)
NE1-1
NE3-1
668.33(+3)->619.32(+2)
CE: 20V
CE: 22V
p12-1
692.94(+2)->859.58(+1)
CE: 20V
CE: 26V
p13-1
363.55(+3)
p16-R*
636.34(+2)->784.38(+1)
CE: 20V
CE: 24V
p18-1
654.00(+3)->597.83(+2)
CE: 18V
CE: 20V
p19-1
672.36(+2)->883.44(+1)
CE: 21V
CE: 22V
p20-1
511.77(+2)->880.46(+1)
CE: 17V
CE: 18V
p21-1
412.22(+2)->595.33(+1)
CE: 13V
CE: 15V
p24-1
569.81(+2)->497.27(+1)
CE: 18V
CE: 22V
p44-1
496.78(+2)->524.29(+1)
CE: 17V
CE: 19V
p45-1
620.33(+3)->823.92(+2)
CE: 16V
CE: 18V
p46-1
657.03(+3)->849.49(+1)
CE: 18V
weak
p47-1
468.57(+3)
pk10-1
534.79(+2)->765.46(+1)
CE: 17V
CE: 20V
pk1-1
550.80(+2)->767.43(+1)
CE: 19V
CE: 21V
pk12-1
527.78(+2)->781.45(+1)
CE: 15V
CE: 18V
pk13-1
798.77(+3)->1027.55(+2)
CE: 24V
weak
pk14-1
618.66(+3)->797.40(+2)
CE: 16V
CE: 17V
pk15-1
461.25(+2)->547.31(+1)
CE: 16V
CE: 19V
pk16-1
485.22(+3)->
pk17-1
635.33(+2)->885.49(+1)
CE: 26V
CE: 28V
pk18-1
851.38(+2)->1007.51(+1)
CE: 28V
CE: 29V
pk19-1
628.62(+3)->707.83(+2)
CE: 18V
weak
pk2-1
287.84(+3)->469.28(+1)
CE: 12V
CE: 14V
pk28-1
492.25(+3)->606.82(+2)
CE: 13V
CE: 15V
pk30-1
582.30(+3)->756.38(+1)
CE: 14V
CE: 16V
pk32-1
677.68(+3)->604.33(+1)
CE: 22V
CE: 22V
pk33-1
703.69(+3)->740.89(+2)
CE: 16V
CE: 16V
pk35-1
626.36(+2)->617.35(+2)
CE: 21V
CE: 20V
pk36-1
314.83(+3)->394.24(+1)
CE: 14V
CE: 15V
pk38-1
545.63(+3)->539.34(+1)
CE: 14V
CE: 16V
pk4-1
552.29(+2)->875.45(+1)
CE: 15V
CE: 18V
pk5-1
363.86(+3)->
pk6-2
454.26(+3)->740.39(+1)
CE: 18V
CE: 20V
pk7-1
559.29(+2)->875.45(+1)
CE: 16V
CE: 18V
pk8-1LK
378.88(+3)->546.30(+1)
CE: 12V
CE: 13V
pk9-1
793.91(+2)->1056.57(+1)
CE: 25V
weak
PRL1-*KK
457.28(+2)->653.39(+1)
CE: 15V
CE: 18V
PRL12-R*
575.79(+2)->858.47(+1)
CE: 19V
CE: 21V
PRL2-*KK
451.26(+2)->653.39(+1)
CE: 15V
CE: 18V
PRL3-F*KK
603.33(+4)->717.39(+3)
CE: 20V
CE: 16V
PRL3-R*
582.80(+2)->872.49(+1)
CE: 18V
CE: 21V
PTP1B-1
594.30(+3)->653.41(+1)
CE: 14V
CE: 18V
PTPRT1
584.67(+3)->483.78(+2)
CE: 14V
CE: 15V
PTPRT2
734.35(+2)
SHP1-1
641.35(+2)->1053.59(+1)
CE: 18V
CE: 22V
SHP1-2
556.30(+2)->463.27(+2)
CE: 14V
CE: 17V
SHP1-3
603.32(+2)->850.44(+1)
CE: 17V
CE: 21V
T10-1
509.31(+2)->690.40(+1)
CE: 15V
CE: 20V
T1-1
360.52(+3)->411.73(+2)
CE: 9V
CE: 12V
T12-1
423.56(+3)->534.78(+2)
CE: 9V
CE: 12V
T15-1
574.31(+2)->672.37(+1)
CE: 19V
CE: 22V
T16-R*
610.78(+2)->628.29(+1)
CE: 19V
CE: 22V
T17-1
443.25(+2)->609.33(+1)
CE: 12V
CE: 16V
T19-1
477.91(+3)->623.82(+2)
CE: 12V
CE: 14V
T2-1
319.83(+3)
T21-1
479.75(+2)->708.47(+1)
CE: 14V
CE: 16V
T22-1
466.58(+3)
T23-1
403.23(+3)->392.27(+1)
weak
T24-1
369.19(+3)
T25-1
587.28(+2)->639.35(+1)
CE: 17V
CE: 20V
T26-1
557.97(+3)->766.44(+1)
CE: 20V
CE: 22V
T27-1
432.91(+3)
T29-1
537.31(+2)->822.51(+1)
CE: 20V
CE: 23V
T30-1
290.52(+2)
T3-1
469.91(+3)->529.26(+1)
CE: 16V
CE: 18V
T31-1
473.26(+3)->624.34(+2)
CE: 13V
CE: 15V
T32-1
563.30(+3)->687.39(+1)
CE: 12V
CE: 15V
T33-1
425.92(+3)->525.30(+2)
CE: 10V
CE: 12V
T35-1
571.98(+3)
T37-1
470.26(+3)
T38-1
458.59(+3)->566.32(+2)
CE: 11V
CE: 13V
T39-1
421.24(+3)->607.36(+1)
CE: 14V
CE: 18V
T40-1
834.43(+2)->906.48(+1)
CE: 27V
CE: 29V
T4-1
663.37(+2)->955.55(+1)
CE: 20V
CE: 24V
T41-1
341.22(+3)->427.29(+2)
CE: 9V
CE: 12V
T45-1
734.91(+2)->1092.64(+1)
CE: 22V
CE: 24V
T46-1
635.34(+2)->1027.55(+1)
CE: 19V
CE: 22V
T47-1
367.21(+3)->441.26(+2)
CE: 8V
CE: 10V
T48-1
497.78(+2)->767.43(+1)
CE: 16V
CE: 19V
T5-1
511.29(+2)->821.46(+1)
CE: 16V
CE: 19V
T53-1
427.57(+3)->540.79(+2)
CE: 14V
T6-1
594.27(+3)
T7-1
489.58(+3)->583.31(+2)
CE: 11V
CE: 14V
T8-1
646.41(+2)->806.52(+1)
CE: 21V
CE: 28V
Energy value of synthetic standard peptide having substitution with an isotope prepared in Example 2 was measured. The synthetic standard peptide has the strongest detection signal, because it preceeded to optimal fragmentation and ionization on tandem mass spectrometer.
2 μl of a mixed sample containing 100 femto mole of each of 87 isotope-substituted synthetic standard peptides (Table 4) was loaded in Q-Tof mass spectrometer (Waters, USA) connected to nanoAquity HPLC, followed by recording the fragmentation pattern with changing energy from 4-30 V by 2 V each time according to Full scan MS/MS method. MS/MS spectrum of each peptide obtained over energy changes was sorted to analyze increase or decrease of fragmented ions. The daughter ion demonstrating the strongest ionic strength and fragmentation energy at that time were recorded. And We confirmed whether theoretically predicted fragmented ion, charge number and mass were consistent If they were consistent, they were finally determined to optimum daughter ion and fragmentation energy of corresponding isotope-substituted standard peptide. The wild type standard peptide had the same molecules and ionic properties with the isotope-substituted synthetic standard peptide. So, fragmentation energy corresponding to ion of the wild type standard peptide corresponding to fragmented ion determined by the isotope-substituted synthetic standard peptide was used.
Mass of the isotope-substituted synthetic standard peptide was presented in “SIS” line of Table 4. Optimum fragmentation energy measured by Q-Tof and Quattro mass spectrometer was presented in “QTOF” and “Quat” lines of Table 4 peptide by peptide. Standard peptide mass and ion number (numbers in parentheses) of the wild type standard peptide (Native) and the isotope-substituted synthetic standard peptide (SIS) were presented in “Native” and “SIS” lines. Mass and ion number of the optimum daughter ion (optimum fragmented peptide) determined by the above method were also presented in “Native” and “SIS” lines. For example, in A(AN)->B(Bn) of “Native” and “SIS” lines of Table 4, A indicates mass of the standard peptide, An indicates ion number of the standard peptide, B indicates mass of the optimum daughter ion generated by the standard peptide and Bn indicates ion number of the optimum daughter ion generated by the standard peptide. “Weak” means weak signal which indicates that no-corresponding value was determined.
Polyclonal antibody binding to the standard peptide was constructed to concentrate the wild type and isotope-substituted standard peptides in sample.
First, a peptide for antigen production was prepared by adding cysteine residue for the purification of an antibody to N-terminal or C-terminal of the standard peptide sequence obtained in Example 2 (Peptron Inc., Korea). Polyclonal antibody binding to the standard peptide was produced by AbFrontier Co., Ltd., Korea using the said standard peptide as an antigen. Particularly, a rabbit was immunized with the above antigen. Three months later, serum of the rabbit was obtained. The standard peptide was loaded on SulfoLink (Pierce, USA) containing iodo-acethyl residue via acetylation of terminal cysteine.
After equilibrium of 1 ml column using equilibrium solution (25 mM Tris-HCl pH8.3, 250 mM NaCl, 0.05% sodium azide; Sigma, USA), 10 ml in of the serum obtained in Example <4-2> was added, followed by antigen-antibody reaction at room temperature with stirring for 2 hours, resulting in anti-standard peptide antibody was conjugated on the column. The column was washed with washing solution (25 mM Tris-HCl pH8.3, 1.0 M NaCl, 0.05% sodium azide) four times, followed by equilibrium again with equilibrium solution. At last, the antibody was eluted using 2.5 and of elution solution (0.2 M glycine, pH 2.5, Sigma, USA).
<5-1> Hydrolysis of Microprotein in Blood
Blood Samples were Provided by 50 Patients Diagnosed with colon cancer, liver cancer and stomach cancer, from which serums were separated. As a normal blood sample, a commercial normal serum mixture (Sigma, USA) was used. The blood samples were centrifuged at 2,000 rpm for 10 minutes. The supernatant serum was stored at −70° C. Proteins (albumin, globulin, etc) dominant in the serum were eliminated by using multiple affinity removal cartridge, Hu-7 kit (Agilent, USA) according to the manufacturer's instruction.
The purified serum was diluted with 0.1 M ammonium bicarbonate, leading to the substitution for trypsin hydrolysis. The serum was then treated with heat or/and denaturant (urea, guanidine-HCl, detergent such as rapigest, etc). Particularly, the sample was treated at 95° C. for 10 minutes or treated with 6-8 M urea or guanidine-HCl and added with RapiGes (final conc: 0.1%), followed by reaction at 60° C. for 2 hours. Then, trypsin was added to the reaction solution at the amount of 1/50-100 plasma protein, followed by reaction at 37° C. for 16 hours. Peptide mixture was obtained by hydrolyzing micro proteins in blood plasma.
<5-2> Extraction of Standard Peptide Using Antibody Column
10-50 femto mole of the isotope-substituted synthetic standard peptide obtained in Example 2 was added to 15 μl of the hydrolyzed peptide mixture in blood plasma obtained in Example <5-1>, followed by incubation at room temperature for 2 hours.
After biotinylation, the anti-standard peptide polyclonal antibody prepared in Example 4 was conjugated to immobilized streptavidin (Pierce, USA). Biotinylation was performed by the following processes; dissolving Sulfo-NHS-LC-Biotin (Pierce, USA) in ultra pure distilled water at the final concentration of 10 mM; mixing target antibody with biotin at the molar ratio of 1:20; and reacting at room temperature for one hour with stirring. Non-reacted biotin was eliminated by dilution with PBS (phosphate buffered saline).
Standard peptide was mixed with the polyclonal antibody conjugated column prepared above, followed by reaction at room temperature for 2 hours. The column was washed with washing solution and 0.1 M ammonium bicarbonate solution 4 times, followed by elution of target peptide using 2% formic acid.
<5-3> Extraction of Standard Peptide Using Antibody Mixture Solution
Antibody mixture solution was used for simultaneous analysis and profiling of multiple standard peptides.
Particularly, 1-10 μg of each antibody against 2-80 standard peptides (rabbit serum or purified antibody) was mixed with peptide mixture (50 mM Tris HCl pH 8.1, 250 mM NaCl) hydrolyzed with trypsin. Reaction was induced at 4° C. for overnight, and then the standard peptide conjugated antibody was concentrated using protein G beads (GE Healthcare, USA). Antibody conjugated beads were washed twice with washing solution, once with 1 M NaCl, and three times with ddH2O, which was mixed with 500 μl of ddH2O, followed by heating at 85° C. for 10 minutes, leading to solubilization of the antibody and peptide from the beads. Standard peptide dissolved in the solution was filtered by using microcon YM10 (Millipore, USA). Standard peptide in flow-through was dried by cold trap type speedvac. Then, the standard peptide was dissolved in 0.1% formic acid, followed by analysis using mass spectrometer.
<6-1> Preparation of Sample for Mass Spectrometry
Desalting from the isotope-substituted synthetic standard peptide obtained in Example 2, the wild type standard peptide obtained in Example <5-2>, and the peptide mixture obtained in Example <5-3> was performed using small rotary column (Waters, USA) filled with C18 column. The peptides were dried by using cold trap type speedvac at 25° C. to −85° C. for 2 and half hours with the pressure of 0.2 torr, which were then dissolved again in 0.1% formic acid solution, leading to analysis using NanoAquity HPLC linked Quattro Premier mass spectrometer (Waters, USA) or the same HPLC linked Q-Tof Premier mass spectrometer. The Quattro Premier mass spectrometer or the Q-Tof Premier mass spectrometer is tandem mass spectrometer, which was used for LC-tandem spectrometry by connecting to NanoAquity HPLC. In particular, Quattro Premier is Triple Quadrupole Mass Spectrometer, which has a high sensitivity, so that it has been largely used for quantitative analysis of peptides in samples. In the meantime, Q-Tof Premier mass spectrometer combining Quadrupole and TOF has also high accuracy in mass analysis and has been largely used for precise measurement of peptide to select standard peptide.
<6-2> Quantitative Analysis Using Tandem Mass Spectrometer
Quantitative analysis was performed with the wild type peptide and the isotope-substituted standard peptide prepared in Example <6-1> for mass analysis by using Quadrupole mass spectrometer which is nanoAQUITY HPLC linked to Quattro Premiere mass spectrometer (Waters, USA). For nanoAQUITY HPLC, BEH300 column (particle size 1.7 μm, ID 75 μm, length 100 mm) was used. The HPLC was operated by density gradient by mixing solution A (0.1% formic acid deionized water) and solution B (0.1% formic acid acetonitrile) with the flow velocity of 300 mL/min for 40 minutes. Capillary voltage of the mass spectrometer was 3.2 kV. For mass spectrometry data collection, MRM (multiple reaction monitoring) routine provided by MassLynx software (Waters, USA) was used. Optimum fragmentation energy of each target standard peptide was used (Table 4) and every isotope-substituted synthetic standard peptide was scanned under the same conditions to analyze exact amount of each peptide. Considering the number of standard peptides to be analyzed at a time, dwell time was adjusted to 0.05-0.02 seconds and interscan time was adjusted to 0.02-0.007 seconds. Absolute quantity of the wild type standard peptide in a sample was determined by comparing chromatogram peaks generated by the isotope-substituted standard peptide whose exact amount added was already known.
As a result, as shown in
<7-1> Composition of PTP Panel
A mixed solution was prepared by mixing 10-50 femto mole of each isotope-substituted synthetic standard peptide prepared in Example 2.
<7-2> Diagnosis of Disease Using PTP Panel
Serums taken from 20-30 cancer patients and normal health people obtained in Example <5-1> were mixed to prepare a mixed serum disease by disease. The mixed serum was treated with trypsin for hydrolysis by the same manner as described in Example <5-1> to obtain a peptide mixture. 10-50 femto mole of the isotope-substituted synthetic standard peptide mixed solution prepared in Example <7-1> was added to 15 μl of the peptide mixture. The wild type standard peptide and the isotope-substituted synthetic standard peptide were concentrated using the standard peptide specific antibody column or the antibody solution mixture by the same manner as described in Example <5-2> or Example <5-3>, followed by desalting by the same manner as described in Example <6-1> and drying in speedvac. The resultant sample was dissolved in 0.1% formic acid, followed by quantification of standard peptide using Quadrupole mass spectrometer which is nanoAquity UPLC linked to Quattro Premiere mass spectrometer, the triple quadrupole mass spectrometer, by the same manner as described in Example <6-2>. Absolute quantity of the wild type standard peptide in a sample was determined by comparing spectrum peaks generated by the isotope-substituted synthetic standard peptide whose exact amount added was already known.
As a result, 18 PTPs were detected from samples of colon cancer, liver cancer and stomach cancer patients in total (Table 5). 12 out of 18 PTPs were only detected in cancer patient samples. 6 PTPs were detected in normal samples as well as in cancer patient samples, but the levels were much higher in cancer patient samples, indicating they can be used for diagnosis of cancer (Table 5). Particularly, three PTPs (T46, pk32 and pk3) were able to be quantified.
TABLE 5
PTP standard peptide found in cancer patient serum (unit: femto mole)
normal
colon
liver
stomach
health
No.
Name
cancer
cancer
cancer
people
1
T18
0.8
1.4
1.0
x
2
pk3
1.0
0.9
x
x
3
T46
3.3
3.4
3.3
x
4
T4
2.5
0.6
x
x
5
pk17
2.7
2.5
2.6
1.2
6
pk32
3.8
2.3
1.4
x
7
T1
4.0
4.4
5.6
3.1
8
T19
2.4
1.6
3.1
1.2
9
T3
10.7
14.3
7.6
ns
10
T41
9.1
7.7
15.6
10.8
11
pk4
ns
ns
4.2
ns
12
T25
ns
ns
28.8
11.1
13
pk12
2.5
0.7
1.3
ns
14
p12
2.4
ns
x
ns
15
p16
1.9
1.0
ns
x
16
p19
x
34.5
ns
ns
17
pk15
x
ns
1.5
x
18
T32
2.2
10.9
ns
5.3
(x, not detected; ns, data is too weak to interpret.)
<7-3> Quantitative Analysis of PTP T46 Standard Peptide
Quantitave analysis was performed with T46 detected in cancer patients but not detected in normal people confirmed in Example <7-2> using serums separated from each disease.
50 femto mole of the isotope-substituted synthetic standard peptide T46 prepared in Example 2 was added to the entire peptide mixture prepared by disease in Example <7-2>. The wild type peptide and the isotope-substituted synthetic standard peptide were concentrated using T46 standard peptide specific antibody and dried by the same manner as described in Example <7-2>. Then, the wild type standard peptide was quantified.
As a result, as shown in
Those skilled in the art will appreciate that the conceptions and specific embodiments disclosed in the foregoing description may be readily utilized as a basis for modifying or designing other embodiments for carrying out the same purposes of the present invention. Those skilled in the art will also appreciate that such equivalent embodiments do not depart from the spirit and scope of the invention as set forth in the appended claims.
Yoon, Tae sung, Hong, Young Joon, Moon, Jeong Hee, Ryu, Seong Eon, Jeong, Dae Gwin, Hong, Seok-II
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| May 25 2010 | RYU, SEONG EON | Korea Research Institute of Bioscience and Biotechnology | ASSIGNMENT OF ASSIGNORS INTEREST SEE DOCUMENT FOR DETAILS | 024495 | /0128 | |
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